Natural vanilla is a complex mixture of flavor components extracted from the beans of vanilla plants, usually Vanilla fragrans. The extraction process involves an initial curing process, during which vanilla-precursor glucosides in the bean break down to form natural vanillin (4-hydroxy-3-methoxybenzaldehyde) and related flavor components, followed by one or more alcohol extractions to remove the relatively hydrophobic flavor components from the bean. Each of these steps may be relatively time-consuming and costly. For example, the curing process typically is carried out by alternately sun-drying and fermenting the beans, followed by additional warehouse curing and dehydration. Total curing times of up to 4 months may be required to obtain the proper flavor and reduce moisture content to prevent molding. Following curing, the beans are crushed for extraction. Best results are obtained when the crushed beans are extracted with a series of progressively more dilute alcoholic solutions. Each extraction requires a minimum of about 5 days.
Because of the relatively high cost involved in growing, harvesting, and extracting vanilla beans, most vanilla flavor "extract" which is sold commercially is synthetic vanillin, made from wood pulp lignin or from clove oil. The synthetic flavor which makes up about 90% of the vanilla flavor market, however, lacks many of the secondary components which contribute to the quality of flavor and aroma of natural vanilla extract.
One potential alternative source of a complex vanilla flavor composition is plant cells or tissue grown in culture (Knuth). The possibility of obtaining secondary plant products, including plant flavor components, from cell or tissue culture has been previously proposed (Staba; Zenk; Dougall; Yeoman; Knuth; and Collin). This approach has been limited, however, by problems of obtaining suitable cell or tissue material for culturing. Studies with a variety of plant types, particularly those which produce essential flavor oils, such as lemon, mint, avocado, and herbs, such as anise, fennel, and sage, indicate that undifferentiated plant tissues are unable to produce the natural oils, possibly because the tissues lack oil glands, and/or because essential metabolic precursors are not produced (Collin). In cultured herb tissues, flavor components reappeared following redifferentiation into roots and shoots (Becker), and in both celery and onion, flavor components reappeared with redifferentiation (Al-Apta; Selby; Turnbull). Partially differentiated callus or suspension cultures from several flavor-producing plants also show synthesis of a variety of flavor components (Nagel; Szoeke; Sugisawa; Sardesai; Kireeva; Tounsley; and Jalal).
Related to this problem is the difficulty of obtaining cell or tissue material that can be expanded readily in culture, produces the secondary products in high yield, and is stable with long-term culturing. In particular, differentiated or partially differentiated cells generally do not grow well in culture. Even where such cells are obtained, the level of secondary product formation may be quite low compared with the natural plant, and therefore poorly competitive with natural flavor extraction methods.
The problem of obtaining flavor material in an easily isolatable form from cell culture has also limited the cell culture approach. In some cases, flavor component(s) may not be secreted from the culture cells, such that the flavor components must be harvested from the cell or tissue material. By way of example, U.S. Pat. No. 3,710,512 describes a method for producing a licorice extract-like material from cultured plant tissue cells suspended in a culture medium. Here the flavor composition is extracted by boiling the culture mix, to release the compound from the cells, filtering the boiled material, to remove cell debris, and concentrating the filtrate. It can be appreciated that product extraction is inefficient both because cells are lost and because the flavor material must be purified from total cell extract material.
In summary, although the possibility of obtaining flavor components in plant cell or tissue culture has been explored, difficulties in obtaining plant cell or tissue material which is capable of (a) growing readily in culture, over long periods, and (b) secreting desired flavor components in a form which allows isolation from the culture medium, have severely constrained this approach.